In the global shadow of emerging multi-drug resistance in many human pathogens, the pipeline of new antibiotics is alarmingly depleted. To address this pressing public health concern, new types of structurally novel molecules that act via new mechanisms must constantly be sought. In the last 25 years, over 50% of all new chemical entities approved as drugs were based on a natural product lead compound. Thus, chemically diverse natural products from diverse, phylogenetically unique organisms are critical leads for the next generation of therapeutic agents. The overarching goal of this proposal is to use microscale chemical and biological techniques to evaluate recently discovered deep-sea vent organisms as a source of unprecedented small molecule natural products with medicinally-relevant anti-infective properties, and will be achieved by the following specific aims: 1) To obtain and chemically extract field-collected and laboratory-cultured deep vent organisms. 2) To chemically profile a library of vent sample HPLC fractions by liquid chromatograph-mass spectrometry (LC-MS) and automated microflow capillary nuclear magnetic resonance (NMR) spectroscopy. 3) To biologically profile the vent sample library for antimicrobial activity in vitro and to test pure compound leads in vitro in bacterial resistance assays and in vivo in rapid, easily performed zebrafish infection models. Deep vent invertebrates and microbial mats collected by deep submergence vehicles will be subjected to small-scale extraction, filtration and HPLC fractionation to assemble a vent sample library of HPLC fractions. Additionally, laboratory cultures of deep vent bacteria subjected to the same protocols will contribute to the sample library. Analytical data including UV absorption, mass spectrometric and NMR spectroscopic data will be obtained for the HPLC fraction library to enable rapid identification of known or potentially new types of chemical compounds. Additional quantities of these HPLC fractions generated by low replicate HPLC runs will be tested in microtiter liquid culture assays against clinically relevant Gram-positive and Gram-negative bacteria. Purified compounds from HPLC fractions with activity in these assays will be submitted for further evaluation against multi-drug resistant bacterial pathogens. In addition, antibacterial pure compound leads will be tested in in vivo models of mycobacterial and staphylococcal infections of embryonic zebrafish. PUBLIC HEALTH RELEVANCE: The treatment of infectious diseases with antibiotics is plagued by the rapid and inevitable development of drug-resistance in the disease causing microorganisms, and requires the constant discovery of new drug leads that produce their antibiotic effect in new ways. This research focuses on the evaluation of unusual, recently discovered deep-sea hydrothermal vent organisms for their production of novel types of natural products for anti-infective drug development. Natural products are the source of the majority of approved antibiotic drugs, and are also a proven source of new types of chemical compounds.